Cellular actin protrusions (e.g. filopodia, microvilli, and stereocilia) display a broad range of lengths and lifetimes critically related to their specific cellular function. Stereocilia, the mechanosensory organelles of hair cells, are a distinctive class of actin-based cellular protrusions with an unparalleled ability to regulate their lengths over time. Our laboratory has made significant advances towards elucidating the mechanisms that underlie the formation, regulation, renewal, and life span of stereocilia. Studies on actin turnover in stereocilia as well as the identification of several deafness-related proteins essential for proper stereocilia structure and function provide new insights into the mechanisms and molecules involved in stereocilia length regulation, long-term maintenance, and potetnial for repair following overstimulation or acoustic trauma. Myosins and their cargo have been implicated in formation and elongation of actin protrusions, but the mechanisms by which they influence F-actin elongation are diverse and not fully understood. Two proteins implicated in inherited deafness, myosin IIIa, a plus end directed motor, and espin1, an actin bundling protein containing an actin-monomer-binding WH2 (WASP homology 2) motif, have been shown to influence the length and shape of mechanosensory stereocilia of the inner ear. Ongoing studies in our lab demonstrate that espin 1, the only isoform of espin that contains ankyrin repeats, shows a spatial and temporal pattern of localization at the tips of stereocilia similar to that described for myosin IIIa, and that the espin 1 ankyrin repeats domain (ARD) interacts with a unique conserved domain in the myosin IIIa carboxyl-terminal tail region. We show that, like myosin IIIa, espin 1 causes stereocilia elongation when overexpressed in cultured hair cells. Using a heterologous expression system we showed an extraordinary elongation of filopodia resulting from the transport of espin 1 to the plus ends of filopodial F-actin by myosin IIIa, and that this elongation is dependent on espin 1 WH2 activity. This study provides the basis for understanding the role myosin IIIa and espin 1 play in regulating stereocilia length, presenting a physiological example where myosins can boost elongation of actin protrusions by transporting actin regulatory factors to the plus ends of actin filaments. This system also demonstrates that the counter action of diffusion by molecular motors may be a general mechanism that can be used by a cell to localize certain components at the distal end of actin protrusions. We are currently also studying myosin IIIb, a shorter myosin III isoform without the C-terminal actin-binding domain. Since there is no explanation why DFNB30 patients with a homozygous mutation in the MYO3A gene have a normal ability to hear for the first twenty years and progressively become deaf from that point and on, we sought answers to whether myosin IIIb compensates for loss of myosin IIIa function. We observed that myosin IIIb is also localized to the same compartment as myosin IIIa and espin 1 in hair cell stereocilia. However, we found that the temporal expression of myosin IIIb differs. Myosin IIIb showed a peak of immunofluorescence around age P2, while myosin IIIa and espin 1 have the peak at age P6. Furthermore, we found that myosin IIIb cannot self-localize to the tips of filopodia due the lack of actin-binding 3THDII in the tail. Myosin IIIb rather depends on espin 1 for the tip localization. Myosin IIIb is also found to down regulate the localization of myosin IIIa to the stereocilia tips of vestibular hair cells but not to the cochlear hair cells. The localization and interplay of myosins IIIa, IIIb, and espin 1 and their influence on stereocilia length shed light on a previously unrecognized molecular complex at the polymerizing end of actin filaments. We postulate that myosin IIIb compensates for myosin IIIa in the function during the development and maturation of stereocilia thus explaining the late onset hearing loss in DFNB30 patients. We also collaborated with Nir Gov from the Weizmann Institute to produce a physical model that describes the active localization of actin-regulating proteins inside stereocilia during steady-state conditions. The mechanism of localization is through the interplay of free diusion and di-rected motion, which is driven by coupling to the treadmilling actin filaments and to myosin motors that move along the actin laments. The resulting localization of both the molecular motors and their cargo is calculated, and is found to have an exponential (or steeper) profile. This localization can be at the base (driven by actin retrograde flow and minus-end myosin motors), or at the stereocilia tip (driven by plus-end myosin motors). The localization of proteins that infuence the actin depolymerization and polymerization rates allow us to describe the narrow shape of the stereocilia base, and the observed increase of the actin polymerization rate with the stereocilia height. Tip links are thought to be an essential element of the mechanoelectrical transduction apparatus in sensory hair cells of the inner ear. In previous structural, histological and biochemical studies in collaboration with Ulrich Mueller (Scripps Institute) we showed that the extracellular domains of two deafness-associated cadherins, cadherin 23 (CDH23) and protocadherin 15 (PCDH15), interact in trans to form the upper and lower part of each tip link, respectively. In a continuation of these collaborative studies using a mouse model for nonsyndromic deafness (DFNB12) called salsa, we were able to show that hearing loss is related to defects in tip links. The phenotype of salsa suggests that DFNB12 is a new class of deafness caused by the loss of the tip links due to an unstable interaction between CDH23 and PCDH15. A DFNB23-causative missense mutation in PCDH15-ECD is also known to affect the interaction between CDH23 and PCDH15 1, suggesting a common athogenesis underlying both DFNB12 and DFNB23. Establishing mouse models for DFNB23 will be important for testing this hypothesis. In the past year, we have also initiated new collaborations with the laboratories of Henrique von Gersdorff of Oregon Health Sciences Universitys Vollum Institute and Jeffrey Diamond of the National Institute of Neurological Disorders and Stroke (NINDS). These collaborations have allowed us to explore the presynaptic distributions of ribbon synapses and their structural relationships with postsynaptic contacts in both hair cells (von Gersdorff collaboration) and bipolar cells of the retina. For the hair cell project, we have been using serial section electron microscopy to generate detailed anatomical measurements of ribbon synapse sizes, distributions, and vesicle pools which will be combined with electrophysiological recordings from the von Gersdorff lab. The bipolar cell project takes a similar approach, by using serial section reconstructions to characterize the synaptic relationship between rod bipolar cells and A17amacrine cells, which contain reciprocal inhibitory synapses back on to the rod bipolar cell terminal, and combining it with functional data from the Diamond lab. Currently, the large-scale reconstruction projects are nearing completion. As a follow-up approach, we plan to use electron tomography and freeze-fracture methods to investigate the structure and composition of individual ribbon synapses.